Solid composition for the oral administration of dyes and diagnostic use thereof

ABSTRACT

The present application discloses solid compositions for the oral administration of dyes, and diagnostic use thereof. Preferably, such diagnostic use is aimed at the diagnostic evaluation of the gastrointestinal tract.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/877,591 filed on Jan. 23, 2018, which in turn is acontinuation of U.S. patent application Ser. No. 15/061,232 filed onMar. 4, 2016, now U.S. Pat. No. 10,265,420, which in turn is acontinuation of U.S. patent application Ser. No. 13/932,321 filed onJul. 1, 2013, now U.S. Pat. No. 9,402,922, which in turn is acontinuation of U.S. patent application Ser. No. 13/602,875 filed onSep. 4, 2012, now U.S. Pat. No. 8,545,811, which in turn is acontinuation-in-part of International Patent Application No.PCT/IB2011/050881 filed on Mar. 2, 2011, which in turn claims priorityto U.S. Provisional Patent Application Ser. No. 61/327,557 filed on Apr.23, 2010 and to Italian Patent Application No. MI2010A000345 filed onMar. 4, 2010. Each of these applications is incorporated herein in itsentirety.

PRIOR ART

The endoscopy is an exceptionally important diagnostic technique for thediagnosis of inflammatory, ulcerative and neoplastic pathologies of thegastrointestinal tract.

Actually, the endoscopy allows observing—from inside the lumen—the stateof preservation and development of the mucosa that covers thegastrointestinal cavity, as well as the surface spraying thereof, thepresence of deformations and/or ulcerations.

More and more powerful and sophisticated endoscope probes have enabledto improve this technique considerably; the progress of the usedmaterials also allowed improving performance in terms of illuminationand resolution power.

More recently there has been an improvement of the conventionaldiagnostic-therapeutic aspects due to the use of image magnification andvital dyes, useful for locally developing a contrasting colour capableof amplifying the resolution diagnostic power of the conventionaltechnique. The use of dyes in endoscopy lead to coining the term“chromoendoscopy” for describing this diagnostic procedure, particularlyuseful for identifying suspicious areas for degenerativecharacteristics.

The use of colouring is generally adopted after completing theendoscopic analysis during the step of withdrawing the endoscopic probe,and after accurately cleaning the mucosa tract to be examined;currently, the dye is applied to the mucosa by spraying a small volumeof a solution averagely concentrated with dye, using a catheter orcapillary pipe directly inserted into the cavity of the endoscopicprobe.

The diffusion of the dye and absorption thereof by the vital cellsmarkedly differentiates the cells with normal vitality from those in theadvanced replication stage, for example characteristic of neoplasticcells.

The dyes usually used are mainly, but not exclusively, the following:methylene blue, congo red, carmine indigo, and/or toluidine blue.

Methylene blue and toluidine blue are uniformly absorbed by the wholeintestinal mucosa, while, in case of an inflammatory process, absorptionthereof by the mucosal cells tends to reduce as the phlogosis worsens.Due to this characteristic, the two dyes are also useful in the step ofremission of the inflammatory processes and in the differentialdiagnosis between pseudopolyps and true polyps. Also carmine indigo hasa similar action, finding application in the long duration inflammatoryforms and with the aim of highlighting carpet lesions, which can containtumoral forms, which are difficult to detect with the conventionalendoscopy in absence of contrasting colour.

Within the procedure for applying the dye, it should be observed thatuse thereof reveals several practical problems that are difficult toresolve due to the considerable application difficulty. First andforemost the pharmacy of the institute where the endoscopy is performedshould be capable of preparing solutions with concentrations generallycomprised between 0.1% and 1% of the dye; then, the endoscopic probeshould be provided with a channel for inserting the capillary catheterwhich carries the solution up to the point of application; then the dyeshould be dispensed uniformly so as to cover the mucosal surface subjectof the evaluation. The need for the simultaneous presence of theseprecise conditions contributes to the difficulty of executing thechromoendoscopy procedure, which is exclusively carried out by the bestdiagnostic centres up to date, with extensive defections by hospitalsand nursing homes specialized in gastroenterology.

Furthermore, it should be taken into account that the use of a solutionto be locally sprayed on the mucosal wall does not entirely solve theproblem regarding forms still latent, in that too small to be detected,as well as the degenerative processes of the digestive system.

Thus, there arises the need of providing a simple and safe use of a dyein diagnostic endoscopies, through a suitable means of administrationalso capable of guaranteeing a homogeneous and complete distribution foran ideal effect of the dye in the area in question.

Now, it has been surprisingly discovered that a solid composition fororal administration allows formulating one, or more, dyes which can thusreach the desired site providing the contrasting image required forendoscopic diagnostic evaluation.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. As the color drawings are being filedelectronically via EFS-Web, only one set of the drawings is submitted.

FIG. 1 shows a colon endoscopic image, obtained during the endoscopy ofa patient who has taken the composition of the invention within the 24hours preceding the endoscopy. This figure shows a non-pathologic,non-coloured endoscopic view.

FIG. 2 shows a colon endoscopic image, obtained during the endoscopy ofa patient who has taken the composition of the invention within the 24hours preceding the endoscopy. The endoscopic view is characterized byan excellent visualization of the details of the mucosa and inparticular of the pattern of crypts, which are thus clear due to theenhancement and reinforcement action of the dye which, depositedhomogeneously, markedly highlights the details of the mucosa that wouldnot be noticeable to the naked eye.

FIG. 3 shows a colon endoscopic image, obtained during the endoscopy ofa patient who has taken the composition of the invention within the 24hours preceding the endoscopy. This view shows that the action of thehomogeneously distributed dye allows a clear distinction between thepathologic areas (red) and the non-pathologic areas (lighter). Suchdistinction would be less marked and clear without the colouringcontrast whose action shows an accurate definition of the details of themucosa and thus an accurate distinction between pathologic andnon-pathologic areas in a clearer manner with respect to the endoscopicvision without the dye.

FIG. 4 shows a colon endoscopic image, obtained during the endoscopy ofa patient who has taken the composition of the invention within the 24hours preceding the endoscopy. This view shows that the action of thehomogeneously distributed dye allows a clear distinction between thepathologic areas (red) and the non-pathologic areas (lighter). Suchdistinction would be less marked and clear without the colouringcontrast whose action shows an accurate definition of the details of themucosa and thus an accurate distinction between pathologic andnon-pathologic areas in a clearer manner with respect to the endoscopicvision without the dye.

FIG. 5 shows that the tablet absorption profile (circles) is markedlydifferent from that of the vial (squares) and it can be classified as atypical absorption profile of a controlled release formulation.

FIG. 6 shows that the tablet absorption profile for the 400 mg tablet(circles) is very similar to the profile obtained with the 200 mg tablet(dots) and, once again, it can be classified as a typical absorptionprofile of a controlled release formulation, also as outlined by thechart B below, where the chart with the dot refers to the 200 mg dosewhile the one with the triangle refers to the 400 mg one.

DESCRIPTION

Thus, the present invention is aimed at providing a solid compositionfor oral administration containing at least one dye suitable for thepreparation and evaluation of the endoscopic diagnostic analysis, and atleast one physiologically acceptable excipient.

Physiologically acceptable excipients according to the invention arepreferably excipients useful for reaching a mass identifiable uniquely,in terms of quality and quantity, and which can be easily administeredthrough the oral cavity.

In particular, the composition of the invention is intended for oraluptake, before being subjected to the endoscopic diagnostic analysis,during or at the end of the procedure for preparing such analysis.

Thus, an object of the present invention is represented by a solidcomposition, for oral administration, containing at least one dye inassociation with at least one physiologically acceptable excipient,intended for allowing early local identification of pathologic forms atthe gastrointestinal mucosa level, preferably precancerous forms ormucosal areas with high inflammation rate.

The composition of the present invention can be instant-release orcontrolled-release type, preferably of the controlled release typecapable of selectively carrying the dye in the regions subject of theanalysis, thus preventing dispersion thereof into areas not subject ofthe analysis.

The expression “instant-release” is used to indicate a compositioncapable of disintegrating quickly and dissolving in the gastric cavity,simultaneously releasing the entire dye contained therein.

The instant-release composition of the invention preferably comprises atleast one dye in association with physiologically acceptable excipients,technologically indispensable to guarantee the quick disintegration anddissolution of the form in the gastric cavity; more preferably, are usedthe so-called super disintegrants, i.e. polymeric substances capable ofswelling on contact with aqueous fluids and triggering a hydrodynamictension within the pharmaceutical form which leads to the breakagethereof into fragments with the ensuing considerable increase of thesurface/volume ratio and, thus, to a more rapid dissolution of the dye/scontained in the administration form.

Suitable super disintegrants are preferably selected from among modifiedstarches, modified celluloses, polymers or cross-linked copolymers (suchas, for example, cross-linked polyvinylpyrrolidone) or mixtures thereof.

The instant-release composition of the invention may also comprise anouter coating, preferably selected from among polymers and copolymers ofthe acrylic or methacrylic acid, alkyl or hydroxyalkyl cellulosederivatives or mixtures thereof.

The possible presence of such an outer coating is useful for avoidingthe coloration of the mucosa of the mouth and/or of the throat duringthe uptake and swallowing by the patient.

The expression “controlled release” of the invention is used to indicatea composition capable of releasing the dye in a selective site-timemanner, i.e. progressive in the areas of interest. Thus, such expressioncomprises the “rapid, delayed or modified” release definition.

The technology suitable for the formulation of controlled releasecomposition of the invention can be selected from among the matrixtechnologies and the reservoir diffusion technologies known in thesector.

Preferably the controlled release solid oral composition of theinvention is formulated according to the multimatrix technologycommercially known under the trade name MMX®, described in theinternational patent applications WO00/76481 and WO00/76478,incorporated herein by reference.

According to a preferred embodiment of the invention, the controlledrelease solid oral composition comprises at least one dye and amultimatrix structure containing:

a) a matrix which consists of lipophilic compounds with melting pointbelow 90° C., and optionally amphiphilic compounds, in which at leastone dye is at least partly incorporated;

b) an outer hydrophilic matrix in which the lipophilic matrix, andoptionally the amphiphilic matrix are dispersed;

c) optionally other physiologically acceptable excipients;

d) optional gastro-resistant coating.

According to a further embodiment, the composition containing at leastone dye comprises:

a) a matrix which consists of lipophilic compounds with melting pointbelow 90° C. and amphiphilic compounds in which said at least one dye isat least partly incorporated;

b) an outer hydrophilic matrix in which the lipophilic/amphiphilicmatrix is dispersed;

c) optionally other physiologically acceptable excipients;

d) optional gastro-resistant coating.

According to another embodiment, the composition containing at least onedye comprises:

a) a matrix which consists of lipophilic compounds with melting pointbelow 90° C., in which said at least one dye is at least partlyincorporated;

b) an outer matrix which consists of hydrophilic compounds andoptionally amphiphilic compounds, in which the lipophilic matrix, isdispersed;

c) optionally other physiologically acceptable excipients;

d) optionally a gastro-resistant coating.

Suitable lipophilic compounds in the present invention are preferablyselected from among saturated, unsaturated or hydrogenated long chainalcohols, saturated or unsaturated or hydrogenated fatty acids, saltsthereof, esters or amides, mono-, di- or triglycerides of fatty acids,polyethoxylated derivatives thereof, waxes, ceramides, cholesterol,cholesterol derivatives or mixtures thereof.

Suitable amphiphilic compounds in the present invention are thenpreferably selected from among polar lipids of type I or II (lecithin,phosphatidylcholine, phosphatidylethanolamine or a mixture thereof),ceramides, glycol alkyl ethers (such as for example, diethylene glycolmonomethyl ether), alkyl sulfate or sulfosuccinate salts or mixturesthereof.

Suitable hydrophilic compounds in the present invention are preferablycompounds forming hydrogel (i.e. compounds which form hydrogel oncontact with aqueous solvents), more preferably selected from amongpolymers or copolymers of the acrylic or methacrylic acid, alkylvinylpolymers, alkyl celluloses, hydroxyalkyl celluloses, carboxyalkylcellulose, modified or plurisubstituted celluloses, polysaccharides,dextrins, pectins, starches, complex starches and starch derivatives,alginic acid, synthetic rubber, natural rubber, polyalcohols or mixturesthereof.

Suitable gastro-resistant coating according to the invention ispreferably selected from among polymers of the acrylic or methacrylicacid, copolymers of the acrylic or methacrylic acid, cellulosederivatives (such as for example cellulose acetate phthalate)hydroxybutyrate-based polymers, shellac or mixtures thereof. Suchgastro-resistant coatings of the invention can also be combined withplasticisers, opacifiers, dyes or mixtures thereof.

While the oral administration of instant-release solid pharmaceuticalforms allows obtaining a coloration of the first portion of thedigestive tract, such as oesophagus or stomach, the administration ofcontrolled release compositions of the invention actually allowsreleasing the dye contained in the composition precisely starting fromthe gastrointestinal segment intended to be subjected to endoscopicevaluation.

Preferably the composition of the present invention is formulated inform of tablet, capsules, granules, microgranules or pellets. Thecapsule form according to the invention may in turn contain granules,microgranules or pellets.

More preferably, the composition of the invention may be formulated inform of gastro-resistant tablet or in form of a capsule containinggranules, microgranules or gastro-resistant pellets.

Furthermore, the composition of the invention may be formulated in adouble layer form, preferably a double layer tablet.

More precisely, instant-release compositions to be administered a fewminutes before carrying out the endoscopy analysis using a glass ofaqueous liquid are preferable for gastroscopy.

The aqueous liquid is used for facilitating the dissolution of thecomposition, thus forming—in loco—a coloured solution that allows thedye to homogeneously reach the mucosa which covers the digestive cavity,and to be absorbed or not by the cells of the mucosa covering thestomach.

In case of gastroscopic analysis, the composition of the invention isthus preferably in form of instant-release tablet or capsule.

The same target may be attained in the small or large intestine, due tothe use of forms of controlled or targeted release oral administrationof the dye; in particular, a composition coated with a thin film ofcontrolled release gastro-resistant polymers is preferred for anenvironmental pH of about 5 for the small intestine.

In case of endoscopic analysis on the small intestine, the compositionof the invention is thus preferably in form of controlled releasecapsule or tablet, with the presence of a gastro-resistant coating morepreferably selected from among mixtures of acrylic and methacryliccopolymers of type A (Eudragit L, or RL, for example).

Even in case of colon endoscopy, it is preferable to use forms of oraladministration in solid oral form, coated with gastro-resistantsubstances, preferably tablet or capsule.

Such gastro-resistant substances are preferably selected from amongacrylic and methacrylic copolymers of type A, type B (such as forexample those commercially known under the trade name Eudragit S or RS),and/or mixtures based on cellulose acetate phthalate, insoluble in anacid environment which become soluble when the pH is neutralized andacquires a value of about 7. A similar event also occurs when theintestinal transit leads the cud to pass through terminal ileum orthrough the ileocecal valve. Obviously, in the latter case, consideringthe fact that the cud takes at least 3-5 hours to complete the transitin the small intestine and an undetermined period of time, ranging froma few minutes to a few hours, for the gastric emptying, theadministration of the dye composition should be carried out suitably inadvance with respect to the endoscopic analysis, generally for a periodcomprised between 4-24 hours, so as to allow the dissolution of the dyein situ, the formation of a concentrated solution within the colon lumendue to the intestinal fluid present therein, and the diffusion of thedye on the mucosa for a period of time in which the endoscopic probe isintroduced into the intestinal cavity.

In order to allow a homogeneous coloration of the luminal membrane ofall colon regions, from the ileocecal area to the ascending, transverse,descending, sigmoid and rectal colon, the release of the dye should notbe instantaneous but progressive and in line with the advancement of thecomposition.

Given that the transit time of the colon tract is once again veryvariable, but estimated at least 8-16 hours, it is clear that acontrolled release composition with the dye being released in vitro inabout 6-8 hours constitutes the best system to allow a homogeneouscoloration of the entire membrane to be analysed endoscopically and,thus, to obtain the best result in terms of diagnostic evaluation.

Useful dyes according to the present invention are preferably selectedfrom among congo red, carmine indigo, methylene blue, toluidine blue ormixtures thereof. However, according to the invention also otherbiocompatible dye substances can be used, as long as they are providedwith a toxicity profile that does not represent an obstacle to oralsystemic administration thereof.

Therefore, the amount of dye that can be used for maximising thestructural contrast of the mucosal cells depends:

-   -   on the inherent capacity of the dye to induce the coloration of        the vital cells,    -   on the period of time that this coloration should be kept in        contact with the cells and    -   on the massive presence of the liquid for washing the mucosa        swallowed during the step of preparing the colonoscopy.

Actually, such parameters may vary the amount of dye from a fewmilligrams to a few grams of substance, divided into one or more solidoral compositions to be swallowed before or during the step of preparingthe endoscopic procedure, or at the end of the procedure.

Preferably, the solid oral composition of the invention comprises atleast one dye in an amount comprised between 10 mg (0.01 g) and 1500 mg(1.5 g), per single composition, more preferably between 50 mg (0.05 g)and 1200 mg (1.2 g) per single composition.

Said at least one dye according to the invention may also be comprisedin an amount between 2 mg (0.002 g) and 1000 mg (1 g), more preferablybetween 10 mg (0.01 g) and 500 mg (0.5 g) per single composition.

Said at least one dye according to the invention may also be comprisedin an amount between 20 mg (0.02 g) and 500 mg (0.5 g), even morepreferably between 25 mg (0.025 g) and 400 mg (0.4 g), per singlecomposition.

According to a preferred embodiment said at least one dye is containedin the solid composition of the invention in an amount equivalent toabout 25 mg (0.025 g).

According to a further embodiment said at least one dye is contained inthe solid composition of the invention in an amount equivalent to about50 mg (0.05 g).

According to another embodiment said at least one dye is contained inthe solid composition of the invention in an amount equivalent to about200 mg (0.2 g).

According to a preferred embodiment of the present invention in case ofgastroscopy the administration is provided for about 30 minutes beforethe execution of one or more compositions of the invention, preferablyinstant-release tablet or capsule.

According to a further embodiment of the present invention, in case ofendoscopy of the small intestine there is provided for theadministration of one or more compositions of the invention, preferablya controlled release tablet protected by a gastro-resistant coating toprevent early dispersion of the dye in the gastric area not intended tobe subjected to the endoscopic evaluation.

According to a further preferred embodiment of the present invention, incase of colonoscopy there is provided for the administration of one ormore compositions of the invention, preferably a controlled releasetablet so as to prevent the dye from being dispersed into areas of thedigestive tract not intended to be subjected to colonoscopy, such as forexample the stomach, duodena and jejunum.

For the preparation of controlled release compositions, one or more dyesare preferably formulated alongside substances capable of impartingprogressive or massive or controlled or prolonged dissolution propertiesto the formulation; in addition, the formulation is coated withsubstances capable of dissolving solely upon reaching a specific pH,generally comprised between 5 and 7, typical of the section subject ofthe intestinal endoscopic evaluation.

Upon reaching the intestinal section of interest, characterised by aspecific pH value at which the gastro-protective coating startsdissolving, it is important that the dissolution of the dye becontrolled in terms of speed so as to ensure that it occurs within thetime indispensable to the intestinal transit, generally comprisedbetween 4 and 24 hours. Various formulation technologies can be usedaccording to the invention for such purpose.

As mentioned previously, the main and known technologies for obtaining acolon release, such as the use of reservoir systems or diffusion orhydrophilic matrix structures, can be applied for preparing thecontrolled release composition of the invention; the multi-matrixtechnology which exploits a sequence of hydrophilic, lipophilic andamphiphilic matrices for obtaining a result as described above is usedin a preferable application of the invention. In a typical applicationof this multimatrix technology, the dye/s is/are first mixed orgranulated with the material capable of forming a lipophilic matrix, inthe presence of one or more amphiphilic substances with surfactantproperties, and lastly this matrix of powders, at any degree ofaggregation, is inserted into a dominant structure formed by polymers orcopolymers of the hydrophilic type, also known as hydrogels, in theanhydrous state or with low residue moisture value.

Alternatively, still according to a typical application of thistechnology, the dye/s should be first mixed or granulated with thematerial capable of forming a lipophilic matrix, and after granulationthis matrix structure, at any degree of aggregation, is inserted into adominant structure formed by polymers or copolymers of hydrophilic typein anhydrous state or with low level of residue humidity in the presenceof one or more amphiphilic substances with surfactant properties andsubsequently the final mixture is subjected to compression.

A gastro-protective coating film, capable of preventing the dissolutionof the tablet in a strongly acid environment, is lastly applied to thesurface of the compositions.

Upon swallowing, such a multimatrix coated composition is protected fromcontact with gastric and intestinal acids up to reaching an environmentwith suitable pH, preferably greater than 5 or 7, where thegastro-protective coating is solubilised and where the dissolutionprogramme—which will lead it to progressively distribute the dyeinserted in the formulation simultaneously with the advancement withinthe digestive cavity—starts.

Furthermore, an object of the present invention is the abovementionedsolid composition for oral administration for diagnostic purpose,preferably in the endoscopic diagnostic evaluation of inflammatory,ulcerative dysplastic, pre-neoplastic and neoplastic pathologies of thegastrointestinal tract, more preferably cancerous or precancerous forms,polyps, pseudopolyps or different inflammatory pathologies of thegastrointestinal tract.

The composition of the present invention is generally applied for oraladministration during the preparatory stage for the gastro-intestinalendoscopic analysis in a single solution or in two or more periods ofadministration. A typical applied administration pattern provides thatthe administration of the composition, preferably tablet, occurs at theend of the preparatory or cleaning stage of the intestinal mucosa,generally carried out through the uptake of purgatives or polyglycoliclaxative substances, or during the preparation procedure, which usuallylasts a few hours.

A further administration pattern provides that the administration of thecomposition of the invention, preferably a tablet, occurs before thepreviously mentioned preparatory or cleaning stage or that saidadministration partly occurs before and partly during such preparatoryor cleaning step.

Lastly, an object of the present invention is a method for performingendoscopic evaluations of the gastrointestinal tract, comprising theadministration, possibly repeated, of the abovementioned composition tobe preferably carried out within the day prior to the endoscopy (i.e.within the preceding 24 hours: preparatory stage), such evaluation beingaimed at the diagnosis of inflammatory, ulcerative pre-neoplastic,dysplastic or neoplastic pathologies of the gastrointestinal tract, morepreferably cancerous or precancerous forms, polyps, pseudopolyps ordifferent inflammatory pathologies of the gastrointestinal tract.

According to a preferred embodiment, the administration of the solidcomposition of the invention occurs, once or repeated over time, before,simultaneously and/or after the uptake of the preparation/cleaningcomposition preceding the endoscopic analysis (for example, but notexclusively, using the drug available in the market by the nameMoviprep®).

The expression cleaning composition mentioned above is used to indicatethe previously mentioned saline, polyglycolic or laxative solution whichis commonly used for cleaning and washing the intestinal mucosa beforethe endoscopic analysis, during the preparatory stage within thepreceding 24 hours.

According to a more preferred embodiment of the invention, the solidcomposition is taken by the subject intending to carry out theendoscopic evaluation of the colon in two administrations where one doseis taken before the washing composition as described above and thesubsequent dose is taken after or during the uptake of the washingcomposition as described previously. According to such embodiment eachdose can be constituted by one, or more, solid compositions of theinvention, preferably one or more tablets with unitary contentcorresponding to a fraction of the entire dose to be administered.

The examples below are meant for clarifying the invention, withoutentailing any restrictions whatsoever with respect thereto.

EXAMPLES Example 1: Instant-Release Coated Tablet for Endoscopy

Description UOM Amt. per tablet Components Methylene blue mg 50.0Lecithin mg 3.0 Stearic acid mg 6.0 Mannitol mg 120.0 Microcrystallinecellulose mg 50.0 Hydroxypropyl cellulose mg 13.0 Sodium starchglycolate mg 4.0 Colloidal hydrated silica mg 2.0 Magnesium stearate mg2.0 Coating Methacrylic acid copolymer type A (Eudragit L) mg 12.0Triethyl citrate mg 1.2 talc mg 5.8 Titanium dioxide mg 3.0

The production process provides for mixing the dye, lecithin, stearicacid and mannitol up to obtaining a homogeneous mixture. Thenmicrocrystalline cellulose, hydroxypropyl cellulose, sodium starchglycolate, colloidal silica are added to the mixture and mixed onceagain. After adding magnesium stearate, the mixture is compressed up toobtaining 250 mg tablets. The tablets are then arranged in a tabletmixer and coated with a methacrylate-based gastro-resistant film andcontaining triethyl citrate as plasticiser in addition to the titaniumdioxide dye and talc, an anti-stick substance. The tablets thus obtainedare subjected to a dissolution test in an acid environment for twohours, where they reveal to be resistant to the dyeing substance. Thetablets yield to the dye within a few minutes upon introduction into aneutral pH environment.

Example 2: Instant-Release Coated Tablet for Endoscopy

Description UOM Amt. per tablet Components Methylene blue mg 600.0Lecithin mg 5.0 Stearic acid mg 10.0 Mannitol mg 340.0 Microcrystallinecellulose mg 123.0 Sodium starch glycolate mg 30.0 Colloidal hydratedsilica mg 20.0 Magnesium stearate mg 12.0 Coating Methacrylic acidcopolymer type A (Eudragit L) mg 30.0 Triethyl citrate mg 3.0 talc mg15.0 Titanium dioxide mg 7.0

The tablet was obtained through the same process indicated in example 1.

The dissolution test applied to the tablet of example 2 allowedestablishing the substantial non-dissolution of the tablets in an acidenvironment with pH at 1 and the subsequent dissolution in vitro of thedye when moved to a 6.8 pH.

Example 3: Instant-Release Coated Tablet for Endoscopy

Description UOM Amt. per tablet Components Methylene blue mg 1200.0Lecithin mg 10.0 Stearic acid mg 20.0 Mannitol mg 200.0 Hydroxypropylcellulose Mg 50.0 Microcrystalline cellulose mg 20.0 Sodium starchglycolate mg 50.0 Colloidal hydrated silica mg 30.0 Magnesium stearatemg 20.0 Coating Methacrylic acid copolymer type A (Eudragit L) mg 40.0Triethyl citrate mg 4.0 talc mg 20.0 Titanium dioxide mg 9.0

The tablet was obtained through the same process indicated in example 1.

Even in this case, the dissolution test applied to the tablets allowedestablishing the substantial non-dissolution of the tablets in an acidenvironment with pH at 1 and the subsequent dissolution in vitro of thedye when moved to a 6.8 pH, mimetic value of the intestinal pH.

Example 4: Intestinal Release Coated Tablet for Endoscopy

Description UOM Amt. per tablet Components Carmine indigo mg 50.0Lecithin mg 3.0 Stearic acid mg 6.0 Mannitol mg 120.0 Microcrystallinecellulose mg 40.0 Hydroxypropyl cellulose mg 23.0 Sodium starchglycolate mg 4.0 Colloidal hydrated silica mg 2.0 Magnesium stearate mg2.0 Coating Methacrylic acid copolymer type A (Eudragit L) mg 6.0Methacrylic acid copolymer type B (Eudragit S) mg 6.0 Triethyl citratemg 1.2 talc mg 5.8 Titanium dioxide mg 3.0

The applied process provides for mixing the dye with the lecithinsurfactant, stearic acid, mannitol and half of the required amount ofmagnesium stearate. After compacting the mixture followed bygranulation, cellulose, sodium starch glycolate, colloidal silica andthe remaining magnesium stearate are added and then, after furthermixing, the final compression is carried out up to obtaining 250 mgtablets. The tablet is then coated with a mixture of methacryliccopolymers of type A and B, so as to extend the resistance to thedissolution in vitro up to a pH≥7, characteristic of the ileocecal andcolon environment.

Example 5: Intestinal Release Coated Tablet for Endoscopy

Description UOM Amt. per tablet Components Carmine indigo mg 500.0Lecithin mg 3.0 Stearic acid mg 6.0 Mannitol mg 120.0 Microcrystallinecellulose mg 50.0 Hydroxyethylcellulose mg 13.0 Sodium starch glycolatemg 4.0 Colloidal hydrated silica mg 2.0 Magnesium stearate mg 2.0Coating Methacrylic acid copolymer type A (Eudragit L) mg 15.0Methacrylic acid copolymer type B (Eudragit S) mg 15.0 Triethyl citratemg 3.0 talc mg 15.0 Titanium dioxide mg 7.0

The applied process provides for mixing the dye with the lecithinsurfactant, stearic acid and mannitol. After homogeneously dispersingthe dye in the mixture, cellulose, sodium starch glycolate, colloidalsilica and the lubricant magnesium stearate are added and then, afterfurther mixing, the final compression is carried out up to obtaining 700mg tablets. The nuclei are then subjected to coating using a mixture ofmethacrylic copolymers of type A and B alongside other auxiliarysubstances: the tablets resist to the dissolution in vitro in an acidenvironment and they dissolve at a pH≥7, characteristic of the ileocecaland colon environment.

Example 6: Intestinal Release Coated Tablet for Endoscopy

Description UOM Amt. per tablet Components Methylene blue mg 50.0Lecithin mg 3.0 Stearic acid mg 6.0 Mannitol mg 120.0 Microcrystallinecellulose mg 35.0 Hydroxypropyl methylcellulose Mg 28.0 Sodium starchglycolate mg 4.0 Colloidal hydrated silica mg 2.0 Magnesium stearate mg2.0 Coating Methacrylic acid copolymer type A (Eudragit L) mg 6.0Methacrylic acid copolymer type B (Eudragit S) mg 6.0 Triethyl citratemg 1.2 talc mg 5.8 Titanium dioxide mg 3.0

The preparation process provides for mixing the dye with lecithin,stearic acid and microcrystalline cellulose, compaction thereof intowafers followed by dry granulation, mixing with the remaining componentsof the nucleus and the final compression to the weight of 250 mg/tab.The coating uses methacrylic derivatives as base and an alcohol solventas application phase.

The tablets thus obtained were subjected to dissolution test in vitro,revealing a good resistance to the acid environment and a progressivetransfer of the dye in the neutral environment with pH at 7.2.

Example 7: A Colon Controlled Release Tablet

Description UOM Amt. per tablet Components Methylene blue mg 200.0Lecithin mg 5.0 Stearic acid mg 14.0 Methylhydroxypropyl cellulose mg180.0 Mannitol mg 140.0 Microcrystalline cellulose mg 140.0 talc mg 10.0Colloidal hydrated silica mg 5.0 Magnesium stearate mg 6.0 CoatingMethacrylic acid copolymer type A (Eudragit L) mg 16.0 Methacrylic acidcopolymer type B (Eudragit S) mg 16.0 Triethyl citrate mg 6.4 talc mg15.6 Titanium dioxide mg 6.0

The composition is obtained through advance mixing and granulation ofthe dye, the lecithin as amphiphilic component, the stearic acid ascomponent of the lipophilic matrix, mannitol and part of the magnesiumstearate; after screening the granules obtained preliminarily, theremaining components and in particular cellulose, capable of producingthe hydrophilic matrix structure are added. The final pharmaceuticalform, obtained by compressing the mixture of powders and granules,weighing about 700 mg, is subjected to coating with a mixture ofcopolymers of methacrylic derivatives of type A and B, supported by aplasticiser, triethyl citrate, by a dye pigment, titanium dioxide, andby an anti-stick agent, such as talc, using ethylic alcohol as solvent.

The tablet thus obtained revealed in vitro a substantial non-dissolutionat acid pH for 2 hours and a progressive dissolution for about 6 hoursin a simulated intestinal medium with 7.2 pH.

Example 8: Colon Endoscope Experimental Test

The same tablet of example 7 was used for carrying out some colonendoscopies in a human being with extremely positive results. A singletablet was administered to a subject about 12 hours before carrying outthe endoscopy, during the intestinal preparation step, followed by theuptake of about further 500 ml of water. The time that elapsed betweenthe uptake of the tablet and the execution of the endoscopy, about 12hours, was useful to allow the tablet to reach the intestinal colonregion and start the progressive and slow transfer of the dye which, dueto the solubilisation in the liquid present therein, allowed thehomogeneous, intense and persistent coloration of the intestinal mucosa.Actually, after the administration the colon environment revealednoticeable areas of coloration, allowing a considerable contrast betweenthe pathologic areas and the normal mucosa which covers the ascending,transverse, descending, sigmoid and rectal colon regions.

FIGS. 1-4 show four (4) colon endoscopic images, obtained during theendoscopy of a patient who has taken the composition of the inventionwithin the 24 hours preceding the endoscopy. The images clearly show howonly some zones of the colon area of the patient are coloured, while theothers are normal. This shows how after the uptake of the composition ofthe invention, the dye highlights solely the pathologic zones of theexamined colon area (see FIGS. 2, 3-4), and not the zones to beconsidered non-pathologic (see FIG. 1).

Thus, this shows the efficiency of the indicated composition whendetermining the elective coloration of the pathologic intestinal areasand, by contrast, a non-pathologic area which, consequently, is free ofcoloration.

Example 8-Bis: Bioavailability Study

The same tablet was used for a bioavailability and pharmacokinetic studyin which the profile of blood absorption and urinary elimination of thedye administered to healthy volunteers during a clinical study of PhaseI was determined; the pharmacokinetic parameters were compared withthose obtained after intravenous administration of a 100 mg dose of dyeand they were as follows:

T max C_(max) AUC_((o-t)) 100 mg i.v. vial 0.10 hrs 2066 ng/ml 11858ng/ml*h 200 mg tablets 16 hrs 1662 ng/ml 32941 ng/ml*h.

The tablets absorption profile is markedly different from that of thevial and it can be classified as a typical absorption profile of acontrolled release formulation, also as shown in FIG. 5.

Example 9: Colon Endoscope Experimental Test

Using the tablet of example 7, there was carried out an endoscopy testof the colon by administering two tablets to a subject awaitingcolonoscopy, according to the specified uptake method, i.e. by takingthe first dye tablet at the end of the preparatory stage and the secondtablet about 6 hours before carrying out the endoscopy evaluation. Thetablets were administered with abundant water, so as to efficientlysupport the in situ dissolution of the tablets.

Also in this case, the coloration was homogeneous and well marked,allowing carrying out the test in optimal conditions for diagnosticpurposes.

Example 9-Bis: Bioavailability Study

The same tablet was used for a bioavailability and pharmacokinetic studyin which the profile of blood absorption and urinary elimination of thedye administered to healthy volunteers during a clinical of Phase I wasdetermined; the pharmacokinetic parameters measured after theadministration of two 200 mg tablets are the following:

T max C_(max) AUC_((0-t)) 16 hrs 1636 ng/ml 38080 ng/ml*h.

The tablets absorption profile is very similar to the profile obtainedwith the 200 mg tablet and, once again, it can be classified as atypical absorption profile of a controlled release formulation, also asshown in FIG. 6, where the chart with the dot refers to the 200 mg dosewhile the one with the triangle refers to the 400 mg one.

Example 10: Colon Release Coated Tablet

Description UOM Amt. per tablet Components Toluidine blue mg 400.0Carmine indigo mg 100.0 Lecithin mg 5.0 Stearic acid mg 10.0 Mannitol mg30.0 Methylhydroxypropyl cellulose mg 95.0 Microcrystalline cellulose mg10.0 Sodium starch glycolate mg 25.0 Colloidal hydrated silica mg 15.0Magnesium stearate mg 10.0 Coating Methacrylic acid copolymer type A(Eudragit L) mg 20.0 Methacrylic acid copolymer type B (Eudragit S) mg20.0 Triethyl citrate mg 4.0 talc mg 20.0 Titanium dioxide mg 9.0

The preparation process provides for the formation of a granulatecontaining toluidine blue, lecithin, stearic acid, mannitol and part ofthe magnesium stearate; after compaction and reduction into granulesthrough screening, the second Carmine indigo dye, cellulose, sodiumstarch glycolate, colloidal silica and the remaining magnesium stearatelubricant are added. After homogenization, the mixture is compressed to700 mg and subsequently subjected to coating as described in example 7,using the two copolymers of the methacrylic acid and the otherfunctional excipients.

The tablet thus obtained resists to dissolution in vitro in buffers withpH<2 and allows a progressive release of the dye substances in bufferswith pH>7.

Example 11: Gastric Immediate Release Coated Tablet

Description UOM Amt. per tablet Components Congo red mg 50.0 Lecithin mg3.0 Stearic acid mg 6.0 Mannitol mg 120.0 Microcrystalline cellulose mg63.0 Sodium starch glycolate mg 4.0 Colloidal hydrated silica mg 2.0Magnesium stearate mg 2.0 Coating Methyl hydroxypropyl cellulose mg 12.0Polyethylene glycol 6000 mg 1.2 talc mg 6.0 Titanium dioxide mg 3.8

Obtained through the direct compression method, followed by coating inan aqueous solvent. The tablet dissolves rapidly in vitro within a fewminutes, according to the specifications required by the regulatoryauthorities for immediate release tablets.

The tablet thus obtained can be used for endoscopic evaluations of thegastroduodenal sector for highlighting local pathologic growths whichcan be related to dysplastic or neoplastic processes still at theinitial stage.

Example 12: Double Layer Tablet

Description UOM Amt. per tablet Layer 1 Congo red mg 100.0 Dioctylsulfosuccinate mg 5.0 Stearic acid mg 10.0 Mannitol mg 100.0Microcrystalline cellulose mg 100.0 Sodium starch glycolate mg 20.0Colloidal hydrated silica mg 10.0 Magnesium stearate mg 5.0 Layer 2Methylene blue mg 100.0 Lecithin mg 7.0 Stearic acid mg 10.0Methylhydroxypropyl cellulose mg 100.0 Mannitol mg 80.0 Microcrystallinecellulose mg 40.0 talc mg 50.0 Colloidal hydrated silica mg 7.0Magnesium stearate mg 6.0 Coating Methyl hydroxypropyl cellulose mg 12.0Polyethylene glycol 6000 mg 1.2 talc mg 6.0 Titanium dioxide mg 3.8

The process provides for mixing the components of layer 1 andcompression thereof, followed by the compression of a mixture of powdersand granules obtained from a previous compaction of some components ofthe layer 2, precisely the dye, lecithin, stearic acid, themicrocrystalline cellulose and mannitol with half of the magnesiumstearate, with the remaining co-formulants.

The tablet, weighing about 850 mg, has two differently coloured distinctlayers formulated for differentially releasing the dye both in thegastric sector and in the subsequent intestinal sector.

Example 13: A Colon Controlled Release Tablet

Description UOM Amt. per tablet Methylene blue mg 25.0 Lecithin mg 3.0Stearic acid mg 10.0 Methylhydroxypropyl cellulose mg 90.0 Mannitol mg121.0 Microcrystalline cellulose mg 140.0 talc mg 3.0 Colloidal hydratedsilica mg 5.0 Magnesium stearate mg 3.0 Coating Methacrylic acidcopolymer type A (Eudragit L) mg 8.0 Methacrylic acid copolymer type B(Eudragit S) mg 8.0 Triethyl citrate mg 3.2 talc mg 7.8 Titanium dioxidemg 3.0

The composition is obtained through advance mixing of the dye, thelecithin as amphiphilic component, the stearic acid as component of thelipophilic matrix, a thirds of the quantity of mannitol; then theremaining components were added and in particular the celluloses,capable of producing the hydrophilic matrix structure up to completionof the formula. The final pharmaceutical form, obtained by compressingthe mixture of powders and granules, unitary weighing of about 320 mg,is subjected to coating with a mixture of copolymers of methacrylicderivatives of type A and B, supported by a plasticiser, triethylcitrate, by a dye pigment, titanium dioxide, by a small quantity of thedye, methylene blue, and by an anti-stick agent, such as talc, usingethylic alcohol as solvent.

The tablet thus obtained revealed in vitro a substantial non-dissolutionat acid pH for 2 hours and a progressive dissolution in a simulatedintestinal medium with 7.2 pH with a release of about 45% within thefirst 4 hours and a release part more than 80% at the eighth hour.

1. A method of detecting a pathology in a subject undergoing colonoscopy, comprising the subject swallowing one or more doses of an extended release tablet prior to the colonoscopy, the tablets comprising: (a) methylene blue; (b) at least one lipophilic compound; and (c) at least one hydrophilic compound; and wherein the tablet is coated with a composition comprising at least one gastro-resistant substance.
 2. The method according to claim 1, wherein the pathology is selected from inflammatory, ulcerative, dysplastic, pre-neoplastic and neoplastic pathologies.
 3. The method according to claim 2, wherein the pathology is a pre-neoplastic pathology or a neoplastic pathology.
 4. The method according to claim 3, wherein the pathology is selected from cancerous forms, pre-cancerous forms, polyps and pseudopolyps.
 5. The method according to claim 1, wherein the extended release tablet comprises 25 mg of methylene blue.
 6. The method according to claim 1, wherein the at least one lipophilic compound is selected from saturated, unsaturated or hydrogenated long chain alcohols, saturated, unsaturated or hydrogenated fatty acids, salts thereof, esters or amides, mono-, di- or triglycerides of fatty acids, polyethoxylated derivatives thereof, waxes, ceramides, cholesterol, and cholesterol derivatives.
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. The method according to claim 1, wherein the at least one hydrophilic compound is selected from polymers or copolymers of the acrylic or methacrylic acid, alkyl vinyl polymers, alkyl celluloses, hydroxyalkyl celluloses, carboxyalkyl celluloses, modified celluloses, plurisubstituted celluloses, polysaccharides, dextrins, pectins, starches, complex starches, starch derivatives, alginic acid, synthetic rubber, natural rubber, and polyalcohol.
 11. The method according to claim 10, wherein the at least one hydrophilic compound is selected from alkyl celluloses, hydroxyalkyl celluloses, carboxyalkyl celluloses, modified celluloses, plurisubstituted celluloses, polysaccharides, dextrins, pectins, and starches.
 12. The method according to claim 11, wherein the at least one hydrophilic compound is selected from alkyl celluloses, hydroxyalkyl celluloses, carboxyalkyl celluloses, and starches.
 13. (canceled)
 14. (canceled)
 15. A method for delivering methylene blue to the colon of a subject, comprising administering to the subject an extended release tablet comprising: (a) methylene blue; (b) at least one lipophilic compound; and (c) at least one hydrophilic compound; and wherein the tablet is coated with a composition comprising at least one gastro-resistant substance.
 16. The method according to claim 15, wherein the extended release tablet comprises 25 mg of methylene blue.
 17. The method according to claim 15, wherein the at least one lipophilic compound is selected from saturated, unsaturated or hydrogenated long chain alcohols, saturated, unsaturated or hydrogenated fatty acids, salts thereof, esters or amides, mono-, di- or triglycerides of fatty acids, polyethoxylated derivatives thereof, waxes, ceramides, cholesterol, and cholesterol derivatives.
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. The method according to claim 15, wherein the at least one hydrophilic compound is selected from polymers or copolymers of the acrylic or methacrylic acid, alkyl vinyl polymers, alkyl celluloses, hydroxyalkyl celluloses, carboxyalkyl celluloses, modified celluloses, plurisubstituted celluloses, polysaccharides, dextrins, pectins, starches, complex starches, starch derivatives, alginic acid, synthetic rubber, natural rubber, and polyalcohol.
 22. The method according to claim 21, wherein the at least one hydrophilic compound is selected from alkyl celluloses, hydroxyalkyl celluloses, carboxyalkyl celluloses, modified celluloses, plurisubstituted celluloses, polysaccharides, dextrins, pectins, and starches.
 23. The method according to claim 22, wherein the at least one hydrophilic compound is selected from alkyl celluloses, hydroxyalkyl celluloses, carboxyalkyl celluloses, and starches.
 24. The method according to claim 23, wherein the at least one hydrophilic compound is selected from hydroxyalkyl celluloses.
 25. (canceled)
 26. (canceled)
 27. The method according to claim 15, wherein the at least one gastro-resistant substance is soluble at a pH of between about 5 and about
 7. 28. The method according to claim 27, wherein the at least one gastro-resistant substance is selected from of polymers of acrylic acid, polymers of methacrylic acid, copolymers of acrylic acid, copolymers of methacrylic acid, cellulose derivatives, hydroxybutyrate-based polymers, and shellac.
 29. The method according to claim 28, wherein the at least one gastro-resistant substance is selected from polymers of acrylic acid, polymers of methacrylic acid, copolymers of acrylic acid, and copolymers of methacrylic acid.
 30. The method according to claim 15, wherein the at least one gastro-resistant substance is selected from methacrylic acid copolymer type A and methacrylic acid copolymer type B. 